While the underlying goal of cancer therapy is to destroy the cancer while avoiding excessive damage to the normal organs of the body, their toxic effects to the body limit present treatments such as chemotherapy and radiation. As such, the maximal tolerable dosage of such therapies is often inadequate to eradicate the tumor. Newer treatment strategies have focused upon identifying antineoplastic agents that can distinguish normal cells from their cancerous counterparts. Oncolytic viruses replicate, spread and selectively destroy cancerous tissue, but are attenuated and do not harm normal tissues. In addition to direct oncolysis, an immune-mediated component contributes to oncolytic virus efficacy in immune-competent mice (i.e., oncolytic viruses have a tumor-vaccination effect mediated at least in part through an anti-tumor CD8+ T cell response). Using immune-competent mice with syngeneic, bilateral subcutaneous (s.c.) tumors, previous studies established that treatment of one tumor with oncolytic virus (HSV-1) induced regression of the treated and untreated contralateral tumor (see Toda M, et al. “Herpes simplex virus as an in situ cancer vaccine for the induction of specific anti-tumor immunity.” Hum Gene Ther 1999; 10:385-93). While treated and untreated tumors both regressed, oncolytic virus was only detected in the treated tumor. Furthermore, regression of the uninjected, contralateral tumor resulted from an anti-tumor CD8+ T-cell response.
However, the current forms of oncolytic viruses are severely limited in their use. Viral replication and spread in normal cells is what causes pathogenesis and must be avoided in oncolytic viruses. There are two general ways of ensuring that a virus does not replicate in normal human tissue. The first way is to identify naturally occurring, non-pathogenic viruses, usually from other animals, that do not replicate efficiently in normal human cells. The second way is to make alterations to pathogenic viruses so that they can no longer replicate efficiently in normal human cells, but do replicate in tumor cells.
Identifying naturally occurring non-pathogenic viruses, especially those from other animals, which do not replicate in normal human cells is generally done by isolating viruses from other animals like rodents or birds and testing the ability of these viruses to infect and replicate in normal human cells as well as human tumor cells. This strategy has identified several viruses such as New Castle Disease Virus, Vesicular Stomatitis Virus, Myxoma Virus, and Seneca Valley Virus, among others, which do not replicate well in normal human cells, but exhibit higher levels of replication and cell killing when they infect human tumor cells. Each of these viruses is a pathogen for a specific lower animal and evolution has finely tuned the genes of these viruses for optimal function in the cells of those animals. For a virus to be successful, functions encoded by viral genes must be compatible with the basic molecular circuitry of the host cell so that they can function to direct synthesis of the viral components necessary to manufacture progeny viruses and spread to uninfected cells. Also, to be successful, the virus must fight off attempts by the infected host cell to block viral replication.
The basic molecular circuitry and anti-viral defense mechanisms of mammalian cells are homologous. This means that a virus adapted for replication in, for example, mouse cells, will likely be able to replicate in human cells, albeit with substantially reduced efficiency because, after all, despite the similarities, there are still big differences between mouse and human cells. For the most part, this ensures that viruses from other animals are not pathogenic to humans. However, because of unrestrained cell growth, tumor cells, compared to normal cells, are often not very good at defending themselves against viruses. This is the basis for using naturally occurring viruses from other animals as anti-cancer agents. These viruses are intrinsically compatible with the molecular circuitry of the human cell and are able to infect and replicate relatively freely in tumor cells because the tumor cell is not very good at blocking the viral replicative program compared to normal cells.
At first glance, the idea of using viruses evolved to efficiently infect and replicate in the normal cells of another animal is a good way to ensure tumor specific replication of an oncolytic virus. However, evolution has shaped these viruses to be very good at replicating in nonhuman cells and their replicative ability is still relatively inefficient in human tumor cells compared to viruses that have co-evolved with humans and are optimized for replication in human cells.
While the idea of using a human virus, optimized through evolution for replication in human cells, is an attractive option for cancer treatment, there is great risk associated with treating humans with a human pathogen precisely because evolution has shaped the human virus to be very good at what it does: infect, replicate in, and kill normal human cells. Therefore, much effort has gone into identifying which genes of human viruses are required for directing viral replication in normal cells, but are not required in tumor cells. This strategy seeks to create mutant human viruses deficient in the genes required for replication in normal cells in order to attenuate the virus, but still maintain the ability to infect, replicate in and kill tumor cells. This strategy has resulted in the creation of adenovirus and Herpes Simplex Virus Type 1 (HSV-1) mutants that do not replicate efficiently in normal human cells and replicate better in many types of human tumor cells. Indeed, the most advanced and clinically successful oncolytic viruses to date are mutant versions of adenovirus (H101—Shanghai Sunway Biotech) and HSV-1 (T-Vec—Amgen), the wildtype versions of which are human viral pathogens.
The ability of an oncolytic virus to replicate efficiently in tumor cells is important, but it is not the only factor affecting therapeutic efficacy. One major obstacle to successful cancer treatment using oncolytic viruses is the adaptive immune system, which is very good at containing viruses at the site of infection by limiting viral spread. The adaptive immune system curtails viral spread by manufacturing antibodies that are able to recognize, bind, and inactivate virus particles before they have a chance to bind to the surface of an uninfected cell and initiate a new round of infection. The adaptive immune system also employs anti-viral CD8+ cytolytic T lymphocytes (CTL), which recognize viral peptides displayed on the infected cell surface by MHC-I. When a CD8+ CTL recognizes a viral peptide displayed by MHC-I, it proceeds to kill the cell. Cell surface display of viral peptides on MHC-I is a key mechanism employed by cells to signal to the immune system that they are infected and should be killed before the virus has been able to synthesize progeny, which are necessary for initiating new rounds of infection and facilitating viral spread from one cell to another.
The adaptive immune system, especially CD8+ CTL, are both friend and foe to oncolytic viruses. They are foes for the reason cited above: CD8+ CTL curtail viral spread and eliminate the virus by killing infected cells, which are the factories assembling the virus particles necessary for sustained viral infection. Limited, transient, oncolytic virus infection that is rapidly cleared minimizes the number of tumor cells killed by the virus and limits therapeutic efficacy. Maximizing the number of tumor cells killed by the virus is important because virally killed tumor cells are excellent substrates for professional antigen presenting cells (pAPC). Virally killed tumor cells are thought to provide tumor associated antigens (TAA) to pAPC and viral replicative intermediates such as double stranded RNA (dsRNA) induce maturation of pAPC and expression of co-stimulatory molecules through Toll like receptor signaling. Mature TAA-load pAPC are able to induce activation and expansion of antitumor CD8+ CTL capable of killing tumor cells that display TAA in complex with MHC-I. These anti-tumor CD8+ CTL are major effectors of anti-tumor immunity and the focus of many immunotherapies that seek to induce the immune system to recognize and eliminate tumor cells throughout the body as if cancer were a simple bacterial or viral infection. Thus, from the viewpoint of an oncolytic virus, there are two types of CD8+ CTL: Anti-viral (Foes) and Anti-tumor (Friends).
The pAPC is a lynchpin in the mechanism of anti-tumor immune induction by oncolytic viruses. Tumor resident pAPC are responsible for processing virally killed cells, displaying TAA, and migrating to the tumor draining lymph node (TDLN) where they induce activation and maturation of anti-tumor CD8+ CTL. Anything that interferes with this ability reduces the efficiency at which anti-tumor immunity is induced by oncolytic virus treatment. Many oncolytic viruses, including Vaccinia, adenovirus, and HSV-1 oncolytic viruses have been shown to infect pAPC and induce the cells to kill themselves through apoptosis. From this perspective, oncolytic viruses are their own worst enemy: they kill the messenger responsible for communicating tumor information to anti-tumor CD8+ CTL.
Thus, there is a strong need to develop new oncolytic viruses that are both safe for the patient and effectively use the patient's own immune system to eliminate tumor cells. In other words, one approach to improving oncolytic viruses is to maximize their ability to spread through tumor tissue in order to kill as many tumor cells as possible and efficiently stimulate friendly immune responses (anti-tumor) while also minimizing the effect enemy immune responses exert on viral replication and spread (anti-viral).
The antigen presentation system (APS) is the major mechanism cells employ to tell the immune system that they are infected with a virus. Specifically, the APS mediates display of cellular and viral peptides in complex with MHC-I at the cell surface. By displaying viral peptides at the cell surface, the infected cell hoists a distinctive flag that tells CD8+ CTL the cell is infected with a virus. If the CD8+ CTL recognizes the viral peptide-MHC-I complex, it will kill the cell in order to stop the virus from continuing to use the cell to produce progeny viruses. The transporter associated with antigen processing (TAP) protein is responsible for carrying peptides from the cytoplasm into the endoplasmic reticulum (ER). Once inside the endoplasmic reticulum, these peptides are loaded on MHC-I for display at the cell surface. One family of viruses, the Herpesviruses, have uniquely evolved functions to inhibit TAP thereby blocking the display of viral peptides, which prevents CD8+ CTL from recognizing and killing virus infected cells. However, to date, only certain members of the herpesvirus family and only one member outside the herpesvirus family, namely cowpox, have been able to evolve TAP inhibitor functions. This selective pressure against evolution of TAP inhibition outside of the herpes family suggests that virally encoded functions that inhibit TAP must have unintended consequences and therefore have been ameliorated by other viral function in order to allow for the permissive evolution of TAP inhibitor function in the viral genome to be of net benefit to the virus's goal of maintaining itself in the host.
It is well established that inhibition of TAP leads to downregulation of peptide-MHC-I complexes from the cell surface (Oosten L E, et al. “TAP-inhibiting proteins US6, ICP47 and UL49.5 differentially affect minor and major histocompatibility antigen-specific recognition by cytotoxic T lymphocytes.” Int Immunol 2007; 19(9): 1115-22). NK cells are known to kill cells that are defective in TAP activity and exhibit low levels of peptide-MHC-I complexes at the cell surface (reviewed in Cassidy S A, et al. “Effects of peptide on NK cell-mediated MHC I recognition.” Front Immunol 2014; 1-8). Clearly, NK cells have the ability to identify cells infected with a virus that encodes a TAP inhibitor. Indeed, a seminal study examined an adenovirus gene therapy vector administered to rhesus macaques. The vector directed expression of the human cystic fibrosis transmembrane regulator (hCFTR) after instillation into the lungs. In addition, it expressed the HSV-1 TAP inhibitor ICP47 with the hope that TAP inhibition would improve the longevity of hCFTR expression by precluding elimination of virally transduced cells by CD8+ CTL that recognize adenoviral antigens displayed by MHC-I. While cells transduced by the vector were resistant to killing by CD8+ CTL, the cells exhibited increased sensitivity to killing by NK cells resulting in no clear improvement in the long term expression of the hCFTR mRNA in transduced rhesus macaque lung tissue (Scaria A., et al. “Adenoviral vector expressing ICP47 inhibits adenovirus-specific cytotoxic T lymphocytes in nonhuman primates.” Mol Ther 2000; 2(5): 505-14). Therefore, any virus encoding a TAP inhibitor must also have a way of dealing with the negative consequences of TAP inhibition, namely by enabling the infected cell to be hidden from killing by NK cells. Therefore, while inserting a TAP inhibitor into a virus that does not naturally encode a TAP inhibitor may be successful in facilitating viral evasion of CD8+ CTL, it may result in increased killing by NK cells and thus have no ultimate benefit to the virus. This may be why so few viral pathogens, outside the herpes family, have found TAP inhibitors useful.
Due to the complex nature of viral genetic networks and the multiple mechanisms the immune system employs to identify and kill virally infected cells, there is no clear consensus as to how to modify oncolytic viruses to mitigate the negative aspects the immune system exerts on oncolytic viruses while simultaneously maximizing the positive effects the immune system exerts on behalf of oncolytic viruses in the killing of uninfected tumor cells.